DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of disease. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability (Knorre et al. (1994) Design and Targeted reactions of Oligonucleotide Derivatives, CRC Press, Boc a Raton, Fla.). Recently, a new class of compound, peptide nucleic acids (PNA) has shown potential as an antisense agent (Nielsen et al Science, 254, 1497-1500, 1991). PNAs comprise nucleic acid mimics in which the sugar-phosphate backbone is replaced with a backbone based on amino acids. PNAs generally exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They are resistant to nuclease and protease attack. Melting temperatures of their duplexes with DNA or RNA are much higher than any of the known DNA compounds, both modified and unmodified. Recently, the solution structure of PNAs has also been determined by nuclear magnetic resonance (Brown et al., Science 265, 777-780, 1994).
The PNAs may be synthesized inexpensively on a large scale. PNAs may be synthesized by either solution phase or solid phase methods adapted from peptide synthesis. For example, PNAs can be synthesized from four protected monomers containing thymidine, cytosine, adenine and guanine via solid-phase peptide synthesis, by a modification of the Merrifield method (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Merrifield, Science 232, 341-347, 1986) employing, for example, BOC-Z protected monomers (Christensen et al., J. Peptide Science 3, 175-183, 1995).
PNAs recognize DNA and RNA in a sequence specific manner and form complexes which can be characterized by biophysical methods. The binding motif is context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiometric 2:1 complexes, whereas PNAs containing both purine and pyrimidine bases afford a 1:1 heteroduplex with mis-match sensitivity comparable to that found in double-stranded (ds)DNA. The 2:1 complexes are formed when a second strand of the PNA binds the major groove of a PNA-DNA duplex through Hoogsteen base pairing. Thus, the triplex is comprised of a PNA/DNA duplex (formed by Watson-Crick hydrogen bonds) with a second PNA strand lying in the major grove of the duplex (held by Hoogsteen hydrogen bonds). These triplex complexes are so stable that "strand invasion" of dsDNA is possible. Binding of the PNA results in formation of a D-loop in the dsDNA. This characteristic is believed useful to manipulate gene expression at the transcriptional level. These 2:1 and 1:1 complexes mediate the antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation (Noble et al., Drug Development Research 34:184-195, 1995). PNAs may be used as antisense or antigene drugs, exploiting the sequence-dependent binding of the PNA portion to single stranded nucleic acids, particularly mRNA, or double-stranded dsDNA, respectively.
Although the biophysical data are very much in favor of the PNAs becoming very successful as nucleic acid binding agents, they suffer from a vital limitation in that they are taken up by cells very poorly.